PCR is a biochemical technique used to amplify, quantify, and identify specific genes related to cancers, infectious diseases, forensics, and hereditary disorders. Exemplary references discussing PCR, which are incorporated by reference in their entirety, include: F. Moltzahn et al., Cancer Res. 2011, 71, 550-560; M. C. Strain et al., PLoS ONE 2013, 8, e55943; P. Liu et al., Lab. Chip 2011, 11, 1041-1048; J. A. Lounsbury et al., Lab. Chip 2013, 13, 1384-1393; and D. Pekin et al., Lab. Chip 2011, 11, 2156-2166. PCR is commonly performed in central laboratory environments and can involve thermocycling of 20-50 μL samples in well plates. The process to complete PCR, including sample preparation, can take several hours and require a number of manual steps. The duration of PCR from sample preparation to DNA analysis can be reduced by integrating laboratory functions in micro total analysis systems.
An important and widely used element of micro total analysis systems is the droplet PCR component. Ultrafast droplet PCR can be performed with integrated heaters, wherein up to 40 thermal cycles can be conducted in less than six minutes. One such reference discussing this process, which is incorporated by reference in its entirety, is P. Neuzil, Nucleic Acids Res. 2006, 34, e77.
In droplet PCR, droplet microfluidics is used to monodispersly portion PCR reaction mixtures into microreactors surrounded by an oil phase. That is, PCR reaction mixtures can be formed as an aqueous droplet, which are surrounded by an oil phase. Typically, the droplets can differ in size, but are typically on the order of nanoliters or picoliters. At the larger end of this size range, DNA is quantified using a cycle threshold calibration curve. DNA has been absolutely quantified using a popular variation of PCR termed “digital PCR” by using picoliter droplets. The initial average DNA copy number per droplet in digital PCR is less than 1, implying a Poisson distribution with 0 or 1 copy in most droplets. One such reference discussing this process, which is incorporated by reference in its entirety, is N. R. Beer et al., Anal. Chem. 2007, 79, 8471-8475. In digital PCR, after thermocycling, only droplets that contain the target DNA fluoresce. The number of fluorescent and nonfluorescent droplets is counted to absolutely quantify the amount of target DNA according to Poisson statistics. Digital PCR is extremely useful and has been shown to quantify extremely rare targets, such as HIV DNA in infected patients undergoing effective treatment. See M. C. Strain et al., supra.
Though impactful, droplet PCR is in need of technical improvement. For example, as discussed in S. L. Angione et al., Anal. Chem., 2012, 84, 2654-2661, which is incorporated by reference in its entirety, microreactors less than microliters in volume are impaired by surface effects. While it is desirable to have an increase in surface area relative to the volume of the droplet for quick transfer, this presents several potential problems. Irreversible adsorption of amphiphilic proteins occurs as the droplet size becomes smaller and the ratio of the surface area to the volume increases. This irreversible adsorption of proteins to hydrophobic interfaces hinders microfluidic assay.
The adsorption of proteins is particularly problematic with droplet PCR, which makes use of Taq polymerase (Taq Pol) to catalyze the reaction. Taq Pol, an enzyme derived from thermophilic bacteria, is especially prone to absorption at hydrophobic interfaces because a large volume fraction of it is hydrophobic. The aliphatic index characterizes the relative hydrophobic volume of a protein and, in general, thermophilic bacterial proteins have large relative hydrophobic volumes to aide in structural stability. The aliphatic index of Taq Pol is 98.6 (compared to the aliphatic index of BSA, which is 76.1). As discussed in F. C. Lawyer et al., Genome Res. 1993, 2, 275-287, which is incorporated by reference in its entirety, Taq Pol is incredibly stable; even at DNA melting temperatures of 95° C., the half-life of Taq Pol is 45 to 50 min. Thus, less stable proteins used in droplet microfluidics would sample a larger structural space, resulting in the interaction with and irreversible denaturing of the less stable proteins on the hydrophobic surface. Thus, adsorption is exacerbated when using unstable proteins in microfluidic droplets.
Many techniques have been employed to overcome the adsorption of proteins, but these methods have several different flaws. As discussed in S. L. Angione et al., Anal. Chem., 2012, 84, 2654-2661, which is incorporated by reference in its entirety, one technique employed to overcome the adsorption of proteins is to increase the Taq Pol concentration in an attempt to replace the adsorbed enzyme. This technique, however, is a wasteful solution that can require up to seven times the concentration of bulk PCR for optimal performance. The technique is further described in F. Wang et al., Biomed. Microdevices 2009, which is incorporated by reference in its entirety.
Another technique, as discussed in A. C. Hatch et al., Lab. Chip 2011, 11, 3838-3845, which is incorporated by reference in its entirety, is to increase the amount of surfactant used in the system. In addition to stabilizing droplets, large amounts of surfactant can competitively bind to the hydrophobic interface and reduce Taq Pol adsorption, though excessive surfactant can inhibit PCR. Similarly, bovine serum albumin may be included in the PCR mixture to competitively bind to the interface. Usually, both methods are employed simultaneously to create an emulsion, but as discussed in F. Diehl et al., Nat. Methods 2006, 3, 551-559 and R. Williams et al., Nat. Methods 2006, 3, 545-550, which are incorporated by reference in their entirety, for successful PCR, the emulsion must be generated on ice. Yet another technique employs the use of fluorinated oils and surfactants with Taq Pol at room temperature to create droplets for digital PCR. This technique, however, requires fluorocarbon specialty chemicals, which increases costs and concerns for the environment.
In view of the above, there is a need for PCR methods, systems, and kits that overcome the issues associated with protein adsorption in microfluidics that do not suffer from the above described flaws.